Interferon plays an important role as a defensive mechanism against virus infections, tumor cell growth and regulation of the immune system. Interferon blocks viral protein synthesis and inhibition of translation in extracts of interferon-treated cells has been correlated with double-stranded RNA (dsRNA)-dependent synthesis of (2-5A) oligonucleotides which activate an endoribonuclease. In addition, the initiation factor eIF-2 is inactivated by dsRNA-dependent phosphorylation. The production of viral dsRNA during the replication cycle of both RNA and DNA containing viruses could be responsible for triggering these interferon-mediated activities. In this proposal we will establish whether the blockage of viral protein synthesis in interferon treated cells infected with a DNA virus is brought about by the activation of the 2-5A polymerase/endonuclease and/or by inactivation eIF-2 by the phosphokinase pathway. We will then determine whether these events are the result of the formation of viral dsRNA during the course of infection. To study the activation of these pathways we will analyze the integrity of viral mRNA in intact interferon-treated cells after infection with vaccinia virus, by translation in a cell-free system, by size estimation, DNA-RNA hybridization and by analysis of both 5' and 3' termini. The role of the phosphokinase will then be examined by analyzing the formation of methionyl tRNA-40S subunit complexes and by phosphorylation of eIF-2. The presence of viral dsRNA will be established by ribonuclease sensitivity, isopycmic centrifugation, electron microscopy and hybridization to viral DNA. A coupled transcription and translation cell-free system activated with vaccinia virus nucleoids will also be used for comparative studies with the intact, infected cells. These experiments will demonstrate whether inhibition of vaccinia virus protein synthesis by interferon is due to degradation of viral mRNA, to a blockage of protein sysnthesis or both.